Diagnostic method for celiac sprue

ABSTRACT

Detection of toxic gluten oligopeptides refractory to digestion and antibodies and T cells responsive thereto can be used to diagnose Celiac Sprue.

In 1953, it was first recognized that ingestion of gluten, a commondietary protein present in wheat, barley and rye causes disease insensitive individuals. Gluten is a complex mixture of glutamine- andproline-rich glutenin and prolamine molecules, which is thought to beresponsible for disease induction. Ingestion of such proteins bysensitive individuals produces flattening of the normally luxurious,rug-like, epithelial lining of the small intestine known to beresponsible for efficient and extensive terminal digestion of peptidesand other nutrients. Clinical symptoms of Celiac Sprue include fatigue,chronic diarrhea, malabsorption of nutrients, weight loss, abdominaldistension, anemia, as well as a substantially enhanced risk for thedevelopment of osteoporosis and intestinal malignancies (lymphoma andcarcinoma). The disease has an incidence of approximately 1 in 200 inEuropean populations.

A related disease is dermatitis herpetiformis, which is a chroniceruption characterized by clusters of intensely pruritic vesicles,papules, and urticaria-like lesions. IgA deposits occur in almost allnormal appearing and perilesional skin. Asymptomatic gluten-sensitiveenteropathy is found in 75 to 90% of patients and in some of theirrelatives. Onset is usually gradual. Itching and burning are severe, andscratching often obscures the primary lesions with eczematization ofnearby skin, leading to an erroneous diagnosis of eczema. Strictadherence to a gluten-free diet for prolonged periods may control thedisease in some patients, obviating or reducing the requirement for drugtherapy. Dapsone, sulfapyridine and colchicines are sometimes prescribedfor relief of itching.

Celiac Sprue is generally considered to be an autoimmune disease and theantibodies found in the serum of the patients support a theory of animmunological nature of the disease. Antibodies to tissuetransglutaminase (tTG) and gliadin appear in almost 100% of the patientswith active CS, and the presence of such antibodies, particularly of theIgA class, has been used in diagnosis of the disease.

The large majority of patients express the HLA-DQ2 [DQ(a1*0501, b1*02)]and/or DQ8 [DQ(a1*0301, b1*0302)] molecules. It is believed thatintestinal damage is caused by interactions between specific gliadinoligopeptides and the HLA-DQ2 or DQ8 antigen, which in turn induceproliferation of T lymphocytes in the sub-epithelial layers. T helper 1cells and cytokines apparently play a major role in a local inflammatoryprocess leading to villous atrophy of the small intestine.

At the present time there is no good therapy for the disease, except tocompletely avoid all foods containing gluten. Although gluten withdrawalhas transformed the prognosis for children and substantially improved itfor adults, some people still die of the disease, mainly adults who hadsevere disease at the outset. An important cause of death islymphoreticular disease (especially intestinal lymphoma). It is notknown whether a gluten-free diet diminishes this risk. Apparent clinicalremission is often associated with histologic relapse that is detectedonly by review biopsies or by increased EMA titers.

Gluten is so widely used, for examples in commercial soups, sauces, icecreams, hot dogs, etc., that patients need detailed lists of foodstuffsto avoid and expert advice from a dietitian familiar with celiacdisease. Ingesting even small amounts of gluten may prevent remission orinduce relapse. Supplementary vitamins, minerals, and hematinics mayalso be required, depending on deficiency. A few patients respond poorlyor not at all to gluten withdrawal, either because the diagnosis isincorrect or because the disease is refractory. In the latter case, oralcorticosteroids (e.g., prednisone 10 to 20 mg bid) may induce response.

Current diagnostic methods for Celiac Sprue are expensive and not veryaccurate. These methods include ELISA-based methods in which eitheranti-gliadin or anti-tTG antibodies in the patient's serum are detectedand in which T cell proliferation upon stimulation with gliadin isobserved. Often, however, these methods are not sensitive enough todetect the diagnostic antibodies in the blood or, as is the case for Tcell proliferation assays, are deemed to be too expensive for routineuse. Typically, even if an individual tests positive in the diagnostictest, the individual must be re-challenged with gliadin (typically aftermaintaining a gluten-free diet for an extended period of time) andexamined by endoscopy, an invasive and often painful procedure.

PCT publication No. WO 01/25793, published 12 Apr. 2001, describespeptides derived from epitope mapping of alpha-gliadin and methods fordiagnosing Celiac Sprue using such peptides. Those methods, however, donot appear to be significantly more sensitive than methods currentlyemployed and so do not overcome the limitations of diagnostic methodscurrently in use.

PCT publication No. WO 02/083722 describes HLA-DQ restricted T cellsreceptors capable of recognizing prolamine-derived peptides involved infood-related immune enteropathy.

There remains a need for better diagnostic methods for Celiac Sprue,methods that are more sensitive than current methods, that do notrequire confirmation by endoscopy, and that do not require that anindividual be challenged with a gluten-containing diet for accuracy. Thepresent invention addresses this need.

SUMMARY OF THE INVENTION

Methods are provided for diagnosing Celiac Sprue, and/or dermatitisherpetiformis, by detecting multivalent toxic gluten oligopeptides in apatient; antibodies that bind to the toxic gluten oligopeptides; or Tcell proliferation elicited by such oligopeptides in a patient.Multivalent toxic gluten oligopeptides have been found to be resistantto cleavage by gastric and pancreatic enzymes, and the presence of suchpeptides results in toxic effects mediated by antibodies and T cellproliferation. By providing methods for detecting the toxic glutenoligopeptides and the toxic effects mediated thereby, improveddiagnostic methods for diagnosing Celiac Sprue are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Brush border membrane catalyzed digestion of theimmunodominant gliadin peptide. FIG. 1A: LC-MS traces of SEQ ID NO:1QLQPFPQPQLPY after digestion with 27ng/μl rat brush border membrane(BBM) protein for the indicated time. Reaction products were separatedby reversed phase HPLC and detected by mass spectroscopy (ion countsm/z=300-2000 g/mol). The indicated peptide fragments were confirmed bycharacteristic tandem MS fragmentation patterns. The SEQ ID NO:2pyroQLQPFPQPQLPY peak corresponds to an N-terminally pyroglutaminatedspecies, which is generated during HPLC purification of the syntheticstarting material. FIG. 1B Abundance of individual digestion products asa function of time. The peptide fragments in FIG. 1A were quantified byintegrating the corresponding MS peak area (m/z=300-2000 g/mol). Theresulting MS intensities are plotted as a function of digestion time(with BBM only, colored bars). The digestion experiment was repeated inthe presence of exogenous DPP IV from Aspergillus fumigatus (ChemiconInternational, CA, 0.28 μU DPP IV/ng BBM protein) and analyzed as above(open bars). The relative abundance of different intermediates could beestimated from the UV₂₈₀ traces and control experiments using authenticstandards. The inserted scheme shows an interpretative diagram of thedigestion pathways of (SEQ ID NO:1) QLQPFPQPQLPY and its intermediates,the BBM peptidases involved in each step, and the amino acid residuesthat are released. The color code for labeling the peptides is similarto that used in A. The preferred breakdown pathway is indicated in bold.APN=aminopeptidase N, CPP=carboxypeptidase P, DPP IV=dipeptidyldipeptidase IV.

FIGS. 2A-2B. C-terminal digestion of the immunodominant gliadin peptideby brush border membrane. FIG. 2A: (SEQ ID NO:3) PQPQLPYPQPQLPY wasdigested by 27 ng/μl brush border membrane (BBM) protein preparationsfor the indicated time and analyzed as in FIG. 1A. The identity of thestarting material and the product (SEQ ID NO:4) PQPQLPYPQPQLP wascorroborated by MSMS fragmentation. The intrinsic mass intensities ofthe two peptides were identical, and the UV₂₈₀ extinction coefficient of(SEQ ID NO:4) PQPQLPYPQPQLP was half of the starting material inaccordance with the loss of one tyrosine. All other intermediates werebelow ≧1%. The scheme below shows the proposed BBM digestion pathway of(SEQ ID NO:3) PQPQLPYPQPQLPY with no observed N-terminal processing(crossed arrow) and the removal of the C-terminal tyrosine bycarboxypeptidase P (CPP) in bold. Further C-terminal processing bydipeptidyl carboxypeptidase (DCP) was too slow to permit analysis of thesubsequent digestion steps (dotted arrows). FIG. 2B: Influence ofdipeptidyl carboxypeptidase on C-terminal digestion. (SEQ ID NO:3)PQPQLPYPQPQLPY in phosphate buffered saline:Tris buffered saline=9:1 wasdigested by BBM alone or with addition of exogenous rabbit lung DCP(Cortex Biochemicals, CA) or captopril. After overnight incubation, thefraction of accumulated SEQ ID NO:4) PQPQLPYPQPQLP (compared to initialamounts of (SEQ ID NO:3) PQPQLPYPQPQLPY at t=0 min) was analyzed as inFIG. 2A, but with an acetonitrile gradient of 20-65% in 6-35 minutes.

FIG. 3. Dose dependent acceleration of brush border mediated digestionby exogenous endoproteases. As seen from FIG. 2A-2B, the peptide (SEQ IDNO:4) PQPQLPYPQPQLP is stable toward further digestion. This peptide wasdigested with 27 ng/μl brush border membranes, either alone, withincreasing amounts of exogenous prolyl endopeptidase (PEP, specificactivity 28 μU/pg) from Flavobacterium meningosepticum (US Biological,MA), or with additional elastase (E-1250, Sigma, MO), bromelain (B-5144,Sigma, MO) or papain (P-5306, Sigma, MO). After one hour, the fractionof remaining (SEQ ID NO:4) PQPQLPYPQPQLP (compared to the initial amountat t=0 min) was analyzed and quantified as in FIG. 1.

FIG. 4. Products of gastric and pancreatic protease mediated digestionof α2-gliadin under physiological conditions. Analysis was performed byLC-MS. The longest peptides are highlighted by arrows and also in thesequence of α2-gliadin (inset).

FIG. 5. In vivo brush border membrane digestion of peptides. LC-UV₂₁₅traces of 25 μM of (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPFbefore perfusion and after perfusion (residence time=20 min). LC-UV₂₁₅traces of 50 μM of (SEQ ID NO:1) QLQPFPQPQLPY before perfusion and afterperfusion (residence time=20 min).

FIG. 6. Alignment of representative gluten and non-gluten peptideshomologous to (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

FIG. 7. Breakdown and detoxification of 33-mer gliadin peptide with PEP.In vitro incubation of PEP (540 mU/ml) with the 33-mer gliadin peptide(100 μM) for the indicated time. In vivo digestion of the 33-mer gliadinpeptide (25 μM) with PEP (25 mU/ml) and the rat's intestine (residencetime=20 min).

DETAILED DESCRIPTION OF THE EMBODIMENTS

Celiac Sprue and/or dermatitis herpetiformis are diagnosed by detectingdigestion-refractory multivalent gluten oligopeptides, antibodies thatbind to such gluten oligopeptides and/or T-cell proliferation producedby such oligopeptides in Celiac Sprue individuals. Gluten oligopeptidesare highly resistant to cleavage by gastric and pancreatic peptidasessuch as pepsin, trypsin, chymotrypsin, and the like. Some of thesepeptides are multivalent, in that they comprise multiple T cell and/orantibody recognition epitopes. The natural covalent linkage of theseepitopes in a polypeptide is a determinant of hyperantigenicity insusceptible individuals, and related to disease development andpathology. By providing for detection of such gluten oligopeptides; ofantibodies specifically reactive thereto; and/or of T-cell proliferationproduced by such oligopeptides in individuals, improved methods ofdiagnosing Celiac Sprue and/or dermatitis herpetiformis are provided.

The present invention arose in part from the discovery of a 33-mergliadin oligopeptide that is refractory to digestion and is a substratefor tTGase. The selectively deamidated 33-mer produced by tTGase actionis a potent activator of T cells. The experimental analyses that led tothe discovery of this 33-mer are described in the Examples below. InExample 1, a variety of immunodominant epitopes (see Arentz-Hansen etal. (2001), J. Exp. Med. 191:603-612) were tested for resistance toproteolytic enzymes encountered in digestion. Based in part on theresults of the experiments of Example 1, an alpha-gliadin was subjectedto similar tests, as described in Example 2. Those tests showed that arelatively large fragment of the gliadin protein was resistant todigestion by intestinal enzymes. This large fragment, which may bereferred to as the 33-mer of the invention, has the sequence (SEQ IDNO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

Multivalent Gluten Oligopeptides

Preferably, antigenic gluten oligopeptides of interest for use in themethods of the invention are multivalent, and comprise multiple T cellor B cell epitopes, usually comprising at least about two epitopes,preferably at least about three epitopes, and where each epitope iseither non-overlapping (i.e., sterically separate) or overlapping. Inother words, a non-overlapping epitope refers to an epitope where theamino acids of a first epitope are not integral to the sequence of asecond epitope and an overlapping epitope refers to an epitope where theamino acids of a first epitope are integral to the sequence of a secondepitope. For oligopeptides comprising non-overlapping eptiopes, eachdistinct epitope is separated from another epitope by at least a peptidebond, and may be separated by one or more amino acids. As used herein,the term “epitope” refers to the portion of an antigen bound by anantibody or T cell receptor, which portion is sufficient for highaffinity binding. In polypeptide antigens, generally a linear epitopefor recognition will be at least about 7 amino acids in length, and maybe 8 amino acids, 9 amino acids, 10 amino acids, or more.

Generally, the oligopeptides comprise a sequence that may be representedby the formula:E₁-X₁-E₂-X₂-E₃. . . X_(n)-E_(y)   (I)

where E₁, E₂ and E₃ are independently selected epitopes, which may bethe same or different including, but not limited to, those having theamino acid sequence: (SEQ ID NO:10) PFPQPQLPY, (SEQ ID NO:18) PQPQLPYPQ,(SEQ ID NO:19) PQLPYPQPQ, (SEQ ID NO:20) PYPQPQLPY, (SEQ ID NO:21)PQPELPYPQ, (SEQ ID NO:22) PFPQPELPY, (SEQ ID NO:23) PQQSFPQQQ, (SEQ IDNO:24) PFPQQPQQPFP, (SEQ ID NO:25) PYPQPELPY, and cons rvativelymodified variants thereof, where X₁ and X₂ are independently selectedspacers, which may be the same or different and comprise a peptide bondor one or more amino acids, where n=0-5, and where y=0-5. If n=0 andy=0, then the oligopeptide comprises the structure: E₁-X₁-E₂-X₂-E₃. Inone embodiment of the invention, the antigenic oligopeptide comprisesSEQ ID NO:12, which has the epitopic structure (where X₁ and X₂ arepeptide bonds): LQLQ PFPQPQLPY PQPQLPYPQ PQLPYPQPQ PF E₁ E₂ E₃Those of skill in the art will understand that additional epitopes(e.g., E₄, E₅, E₆, etc.), each separated by an additional peptide bondor one or more amino acids (e.g., X₃, X₄, X₅, etc.), are within thescope of the present invention.

Alternatively, the oligopeptides comprise at least one epitope thatoverlaps with at least one other epitope. As such, in another embodimentof the present invention, E₁ and E₂ and/or E₂ and E₃ of Formula I arenot separated by spacers such as X₁ and X₂, but instead contain at leastone overlapping amino acid, preferably at least two or three aminoacids, and more preferably at least four amino acids. Suitableoverlapping epitopes include, but are not limited to, those having theamino acid sequence: (SEQ ID NO:10) PFPQPQLPY, (SEQ ID NO:18) PQPQLPYPQ,(SEQ ID NO:19) PQLPYPQPQ, (SEQ ID NO:20) PYPQPQLPY, (SEQ ID NO:21)PQPELPYPQ, (SEQ ID NO:22) PFPQPELPY, (SEQ ID NO:23) PQQSFPQQQ, (SEQ IDNO:24) PFPQQPQQPFP, (SEQ ID NO:25) PYPQPELPY, and conservativelymodified variants thereof. Those of skill in the art will understandthat oligopeptides comprising a combination of non-overlapping andoverlapping epitopes (e.g., E₁-X₁-E₂-E₃, E₁-E₂-X₂-E₃, etc.) are withinthe scope of the present invention. For example, the antigenicoligopeptide can comprise SEQ ID NO:12, which has the epitopic structureE₁-X₁-E₂-E₃ (where E₂ is PQPQLPYPQ, E₃ is PYPQPQLPY, and E₂ and E₃contain a four amino acid overlap, indicated in bold): LQLQ PFPQPQLPYPQPQLPYPQPQLPY PQPQPF E₁ E₂-E₃

While any combination of the elements comprising E₁, E₂, and E₃ maycomprise the oligopeptides of the present invention, certaincombinations are preferred. For example, for oligopeptides containingnon-overlapping epitopes, wherein the epitopes are selected from thegroup consisting of (1) (SEQ ID NO:10) PFPQPQLPY, (2) (SEQ ID NO:18)PQPQLPYPQ, (3) SEQ ID NO:19) PQLPYPQPQ, (4) (SEQ ID NO:20) PYPQPQLPY,(5) (SEQ ID NO:21) PQPELPYPQ, (6) (SEQ ID NO:22) PFPQPELPY, (7) (SEQ IDNO:23) PQQSFPQQQ, (8) (SEQ ID NO:24) PFPQQPQQPFP, and (9) (SEQ ID NO:25)PYPQPELPY, the following oligopeptides or conservatively modifiedvariants thereof are preferred: 1; 1; 1 1; 1; 2 1; 1; 3 1; 1; 4 1; 1; 51; 1; 6 1; 1; 7 1; 1; 8 1; 1; 9 1; 2; 1 1; 3; 1 1; 4; 1 1; 5; 1 1; 6; 11; 7; 1 1; 8; 1 1; 9; 1 2; 1; 1 3; 1; 1 4; 1; 1 5; 1; 1 6; 1; 1 7; 1; 18; 1; 1 9; 1; 1 2; 2; 1 2; 2; 2 2; 2; 3 2; 2; 4 2; 2; 5 2; 2; 6 2; 2; 72; 2; 8 2; 2; 9 2; 3; 1 2; 3; 2 2; 3; 3 2; 3; 4 2; 3; 5 2; 3; 6 2; 3; 72; 3; 8 2; 3; 9 2; 4; 1 2; 4; 2 2; 4; 3 2; 4; 4 2; 4; 5 2; 4; 6 2; 4; 72; 4; 8 2; 4; 9 2; 5; 1 2; 5; 2 2; 5; 3 2; 5; 4 2; 5; 5 2; 5; 6 2; 5; 72; 5; 7 2; 5; 9 2; 6; 1 2; 6; 2 2; 6; 3 2; 6; 4 2; 6; 5 2; 6; 6 2; 6; 72; 6; 8 2; 6; 9 2; 7; 1 2; 7; 2 2; 7; 3 2; 7; 4 2; 7; 5 2; 7; 6 2; 7; 72; 7; 8 2; 7; 9 2; 8; 1 2; 8; 2 2; 8; 3 2; 8; 4 2; 8; 5 2; 8; 6 2; 8; 72; 8; 8 2; 8; 9 2; 9; 1 2; 9; 2 2; 9; 3 2; 9; 4 2; 9; 5 2; 9; 6 2; 9; 72; 9; 8 2; 9; 9 3; 2; 1 3; 2; 2 3; 2; 3 3; 2; 4 3; 2; 5 3; 2; 6 3; 2; 73; 2; 8 3; 2; 9 3; 3; 1 3; 3; 2 3; 3; 3 3; 3; 4 3; 3; 5 3; 3; 6 3; 3; 73; 3; 8 3; 3; 9 3; 4; 1 3; 4; 2 3; 4; 3 3; 4; 4 3; 4; 5 3; 4; 6 3; 4; 73; 4; 8 3; 4; 9 3; 5; 1 3; 5; 2 3; 5; 3 3; 5; 4 3; 5; 5 3; 5; 6 3; 5; 73; 5; 8 3; 5; 9 3; 6; 1 3; 6; 2 3; 6; 3 3; 6; 4 3; 6; 5 3; 6; 6 3; 6; 73; 6; 8 3; 6; 9 3; 7; 1 3; 7; 2 3; 7; 3 3; 7; 4 3; 7; 5 3; 7; 6 3; 7; 73; 7; 8 3; 7; 9 3; 8; 1 3; 8; 2 3; 8; 3 3; 8; 4 3; 8; 5 3; 8; 6 3; 8; 73; 8; 8 3; 8; 9 3; 9; 1 3; 9; 2 3; 9; 3 3; 9; 4 3; 9; 5 3; 9; 6 3; 9; 73; 9; 8 3; 9; 9 4; 2; 1 4; 2; 2 4; 2; 3 4; 2; 4 4; 2; 5 4; 2; 6 4; 2; 74; 2; 8 4; 2; 9 4; 3; 1 4; 3; 2 4; 3; 3 4; 3; 4 4; 3; 5 4; 3; 6 4; 3; 74; 3; 8 4; 3; 9 4; 4; 1 4; 4; 2 4; 4; 3 4; 4; 4 4; 4; 5 4; 4; 6 4; 4; 74; 4; 8 4; 4; 9 4; 5; 1 4; 5; 2 4; 5; 3 4; 5; 4 4; 5; 5 4; 5; 6 4; 5; 74; 5; 8 4; 5; 9 4; 6; 1 4; 6; 2 4; 6; 3 4; 6; 4 4; 6; 5 4; 6; 6 4; 6; 74; 6; 8 4; 6; 9 4; 7; 1 4; 7; 2 4; 7; 3 4; 7; 4 4; 7; 5 4; 7; 6 4; 7; 74; 7; 8 4; 7; 9 4; 8; 1 4; 8; 2 4; 8; 3 4; 8; 4 4; 8; 5 4; 8; 6 4; 8; 74; 8; 8 4; 8; 9 4; 9; 1 4; 9; 2 4; 9; 3 4; 9; 4 4; 9; 5 4; 9; 6 4; 9; 74; 9; 8 4; 9; 9 5; 2; 1 5; 2; 2 5; 2; 3 5; 2; 4 5; 2; 5 5; 2; 6 5; 2; 75; 2; 8 5; 2; 9 5; 3; 1 5; 3; 2 5; 3; 3 5; 3; 4 5; 3; 5 5; 3; 6 5; 3; 75; 3; 8 5; 3; 9 5; 4; 1 5; 4; 2 5; 4; 3 5; 4; 4 5; 4; 5 5; 4; 6 5; 4; 75; 4; 8 5; 4; 9 5; 5; 1 5; 5; 2 5; 5; 3 5; 5; 4 5; 5; 5 5; 5; 6 5; 5; 75; 5; 8 5; 5; 9 5; 6; 1 5; 6; 2 5; 6; 3 5; 6; 4 5; 6; 5 5; 6; 6 5; 6; 75; 6; 8 5; 6; 9 5; 7; 1 5; 7; 2 5; 7; 3 5; 7; 4 5; 7; 5 5; 7; 6 5; 7; 75; 7; 8 5; 7; 9 5; 8; 1 5; 8; 2 5; 8; 3 5; 8; 4 5; 8; 5 5; 8; 6 5; 8; 75; 8; 8 5; 8; 9 5; 9; 1 5; 9; 2 5; 9; 3 5; 9; 4 5; 9; 5 5; 9; 6 5; 9; 75; 9; 8 5; 9; 9 6; 2; 1 6; 2; 2 6; 2; 3 6; 2; 4 6; 2; 5 6; 2; 6 6; 2; 76; 2; 8 6; 2; 9 6; 3; 1 6; 3; 2 6; 3; 3 6; 3; 4 6; 3; 5 6; 3; 6 6; 3; 76; 3; 8 6; 3; 9 6; 4; 1 6; 4; 2 6; 4; 3 6; 4; 4 6; 4; 5 6; 4; 6 6; 4; 76; 4; 8 6; 4; 9 6; 5; 1 6; 5; 2 6; 5; 3 6; 5; 4 6; 5; 5 6; 5; 6 6; 5; 76; 5; 8 6; 5; 9 6; 6; 1 6; 6; 2 6; 6; 3 6; 6; 4 6; 6; 5 6; 6; 6 6; 6; 76; 6; 8 6; 6; 9 6; 7; 1 6; 7; 2 6; 7; 3 6; 7; 4 6; 7; 5 6; 7; 6 6; 7; 76; 7; 8 6; 7; 9 6; 8; 1 6; 8; 2 6; 8; 3 6; 8; 4 6; 8; 5 6; 8; 6 6; 8; 76; 8; 8 6; 8; 9 6; 9; 1 6; 9; 2 6; 9; 3 6; 9; 4 6; 9; 5 6; 9; 6 6; 9; 76; 9; 8 6; 9; 9 7; 2; 1 7; 2; 2 7; 2; 3 7; 2; 4 7; 2; 5 7; 2; 6 7; 2; 77; 2; 8 7; 2; 9 7; 3; 1 7; 3; 2 7; 3; 3 7; 3; 4 7; 3; 5 7; 3; 6 7; 3; 77; 3; 8 7; 3; 9 7; 4; 1 7; 4; 2 7; 4; 3 7; 4; 4 7; 4; 5 7; 4; 6 7; 4; 77; 4; 8 7; 4; 9 7; 5; 1 7; 5; 2 7; 5; 3 7; 5; 4 7; 5; 5 7; 5; 6 7; 5; 77; 5; 8 7; 5; 9 7; 6; 1 7; 6; 2 7; 6; 3 7; 6; 4 7; 6; 5 7; 6; 6 7; 6; 77; 6; 8 7; 6; 9 7; 7; 1 7; 7; 2 7; 7; 3 7; 7; 4 7; 7; 5 7; 7; 6 7; 7; 77; 7; 8 7; 7; 9 7; 8; 1 7; 8; 2 7; 8; 3 7; 8; 4 7; 8; 5 7; 8; 6 7; 8; 77; 8; 8 7; 8; 9 7; 9; 1 7; 9; 2 7; 9; 3 7; 9; 4 7; 9; 5 7; 9; 6 7; 9; 77; 9; 8 7; 9; 9 8; 2; 1 8; 2; 2 8; 2; 3 8; 2; 4 8; 2; 5 8; 2; 6 8; 2; 78; 2; 8 8; 2; 9 8; 3; 1 8; 3; 2 8; 3; 3 8; 3; 4 8; 3; 5 8; 3; 6 8; 3; 78; 3; 8 8; 3; 9 8; 4; 1 8; 4; 2 8; 4; 3 8; 4; 4 8; 4; 5 8; 4; 6 8; 4; 78; 4; 8 8; 4; 9 8; 5; 1 8; 5; 2 8; 5; 3 8; 5; 4 8; 5; 5 8; 5; 6 8; 5; 78; 5; 8 8; 5; 9 8; 6; 1 8; 6; 2 8; 6; 3 8; 6; 4 8; 6; 5 8; 6; 6 8; 6; 78; 6; 8 8; 6; 9 8; 7; 1 8; 7; 2 8; 7; 3 8; 7; 4 8; 7; 5 8; 7; 6 8; 7; 78; 7; 8 8; 7; 9 8; 8; 1 8; 8; 2 8; 8; 3 8; 8; 4 8; 8; 5 8; 8; 6 8; 8; 78; 8; 8 8; 8; 9 8; 9; 1 8; 9; 2 8; 9; 3 8; 9; 4 8; 9; 5 8; 9; 6 8; 9; 78; 9; 8 8; 9; 9 9; 2; 1 9; 2; 2 9; 2; 3 9; 2; 4 9; 2; 5 9; 2; 6 9; 2; 79; 2; 8 9; 2; 9 9; 3; 1 9; 3; 2 9; 3; 3 9; 3; 4 9; 3; 5 9; 3; 6 9; 3; 79; 3; 8 9; 3; 9 9; 4; 1 9; 4; 2 9; 4; 3 9; 4; 4 9; 4; 5 9; 4; 6 9; 4; 79; 4; 8 9; 4; 9 9; 5; 1 9; 5; 2 9; 5; 3 9; 5; 4 9; 5; 5 9; 5; 6 9; 5; 79; 5; 8 9; 5; 9 9; 6; 1 9; 6; 2 9; 6; 3 9; 6; 4 9; 6; 5 9; 6; 6 9; 6; 79; 6; 8 9; 6; 9 9; 7; 1 9; 7; 2 9; 7; 3 9; 7; 4 9; 7; 5 9; 7; 6 9; 7; 79; 7; 8 9; 7; 9 9; 8; 1 9; 8; 2 9; 8; 3 9; 8; 4 9; 8; 5 9; 8; 6 9; 8; 79; 8; 8 9; 8; 9 9; 9; 1 9; 9; 2 9; 9; 3 9; 9; 4 9; 9; 5 9; 9; 6 9; 9; 79; 9; 8 9; 9; 9.

For example, the structure of oligopeptide 1;1;1 is as follows:PFPQPQLPY PFPQPQLPY PFPQPQLPY. (SEQ ID NO:26)In the foregoing manner, each of the remaining oligopeptides listedabove is described to the same extent as oligopeptide 1;1;1 has beendescribed.

In a further embodiment, the oligopeptides of the present inventioncontain “flanking sequences,” which herein refer to sequences comprisingat least one amino acid at the amino terminus and/or carboxyl terminusof the oligopeptide that is not an epitope. As such, oligopeptides ofthe present invention can contain flanking sequences comprising one,two, three, four, or more amino acids at the amino terminus and/or atthe carboxyl terminus, as long as the flanking sequences are notepitopes.

Other oligopeptides of the invention useful in the methods of theinvention include oligopeptides having the following sequences, andfragments thereof: (SEQ ID NO:13) QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ(from α1- and α6-gliadins); (SEQ ID NO:14)QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein); (SEQ ID NO:15)QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ; (SEQ ID NO:16)PQQPQLPFPQQPQQPFPQPQQPQQPFPQSQQPQQPFPQPQQQFPQPQQPQQSFPQQQQ P (fromγ-gliadin); and (SEQ ID NO:17) QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ. Theseoligopeptides are resistant toward endo- and exo-proteolysis by gastric,pancreatic and small intestinal enzymes, comprise multiple epitopes, andare recognized by tTGase. See, for example, Molberg et al, (1998) Nat.Med.; Vader et al, (2002) J. Exp. Med.; Sollid, et al. (2000) Ann. Rev.Immunol 2000; Vader et al, (2003) Gastroenterology; and Osman et al(2000) Clin. Exp. Immunol. 121, 248-254.

Antibodies may also recognize conformational determinants formed bynon-contiguous residues on an antigen, and an epitope can thereforerequire a larger fragment of the antigen to be present for binding, e.g.a protein domain, or substantially all of a protein sequence. Thebinding site of antibodies typically utilizes multiple non-covalentinteractions to achieve high affinity binding. While a few contactresidues of the antigen may be brought into close proximity to thebinding pocket, other parts of the antigen molecule can also be requiredfor maintaining a conformation that permits binding. In order toconsider an antibody interaction to be “specific”, the affinity will beat least about 10⁻⁷ M, usually about 10⁻⁸ M to 10⁻⁹ M, and may be up to10⁻¹¹ M or higher for the epitope of interest. It will be understood bythose of skill in the art that the term “specificity” refers to such ahigh affinity binding, and is not intended to mean that the antibodycannot bind to other molecules as well. One may find cross-reactivitywith different epitopes, due, e.g. to a relatedness of antigen sequenceor structure, or to the structure of the antibody binding pocket itself.

The T cell receptor recognizes a more complex structure than antibodies,and requires both a major histocompatibility antigen binding pocket andan antigenic peptide to be present. The binding affinity of T cellreceptors is lower than that of antibodies, and will usually be at leastabout 10⁻⁴ M, more usually at least about 10⁻⁵ M.

Affinity and stability are different measures of binding interaction.The definition of affinity is a thermodynamic expression of the strengthof interaction between a single antigen binding site and a singleantigenic determinant (and thus of the stereochemical compatibilitybetween them). Affinity does not change with valency, because it is themeasure of interaction between a single binding site and a singleantigenic determinant. In contrast to affinity, avidity (which relatesto the t_(1/2) of an interaction) is defined as the strength of binding,usually of a small molecule with multiple binding sites by a largermolecule, and in particular, the binding of a complex antigen by anantibody. Therefore, it is avidity that takes into account the effect ofmultiple interactions, and it is the change in avidity that may providesthe hyperantigenicity observed with the oligopeptide of SEQ ID NO:12.

It is also shown herein that the 33-mer (SEQ ID NO:12) is a particularlygood substrate for the enzyme tTGase, which deamidates the 33-mer atleast at the underlined positions shown in the following sequence: (SEQID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF.

Antigenic oligopeptides of the present invention may comprise deamidatedglutamine residues at one, two, three or more positions, which positionsmay or may not correspond with those of the naturally deamidatedoligopeptides.

The 33-mer (SEQ ID NO:12) and its deamidated counterparts have beentested for an ability to stimulate monoclonal and polyclonal T celllines from Celiac Sprue individuals. Their stimulatory ability wascompared with that of a number of immunogenic epitopes contained inshorter peptides, and the 33-mer and its deamidated counterparts wereshown to be far more specific and potent than the shorter peptides.

These results provide the basis for a number of improved diagnosticmethods for Celiac Sprue as well as a variety of reagents useful inthose and other methods. The multivalent gluten oligopeptides describedherein, including those comprising SEQ ID NO:12; deamidatedcounterparts, derivatives, analogs, and conservatively modified variantsthereof, are useful in stimulating T cells from Celiac Sprue patientsfor diagnostic purposes, and so are provided by the present invention inisolated and highly purified forms. Further, the multivalent glutenoligopeptides described herein, including those comprising SEQ ID NO:12;deamidated counterparts, derivatives, analogs, and conservativelymodified variants thereof, are useful in diagnostic assays for detectingantibodies against such oligopeptides or for producing antibodies thatbind specifically to such oligopeptides for their detection.

Transglutaminase Oligopeptide Fusions

In one embodiment of the invention, a fusion protein comprising all or aportion of a mammalian tTGase, including but not limited to human,bovine, equine, and porcine tTGase, is linked, usually covalently, to amultivalent gluten oligopeptide of the invention, wherein the linkagesite is at a site for eventual deamidation. This fusion protein of theinvention is a highly potent stimulator of T cells from Celiac Spruepatients in that the fusion protein exactly mimics the complexes formedin Celiac Sprue patients and is recognized by the anti-tTGase antibodiesand by T cells in those patients. Such fusion proteins find use in thediagnostic methods of the invention.

Transglutaminases (EC 2.3.2.13) are a family of enzymes that catalyzethe crosslinking of proteins by epsilon-gamma glutamyl lysine isopeptidebonds. The human haploid genome contains at least 8 distincttransglutaminases that are differentially expressed in time-space andtissue-specific ways, and these enzymes find use in the presentinvention. Although the overall primary structures of these enzymesappear to be quite different, they all share a common amino acidsequence at the active site (Y-G-Q-C-W) and a strict calcium dependencefor their activity. The differences in the primary structures of thesedifferent transglutaminases are responsible for the diverse biologicfunctions that they play in physiologic processes.

Transglutaminases of particular interest include the human TG1, TG2 andTG3 enzymes. Keratinocyte transglutaminase, TG1, has the Genbankaccession number D90287 (see Phillips et al. (1990) Proc. Natl. Acad.Sci. U.S.A. 87(23):9333-9337; Yamanishi et al. (1991) Biochem. Biophys.Res. Commun. 175(3):906-913). It is normally expressed in skin, and isinvolved in the barrier formation of keratinocytes. The human proteinhas a molecular mass of about 90 kD, having a 105-residue extensionbeyond the N terminus of the tissue transglutaminase (TG2). The deduced813-amino acid sequence of the TG1 protein shares 49 to 53% homologywith other transglutaminase proteins of known sequence.

Tissue transglutaminase 2 (TG2) has the Genbank accession number M55153,and encodes a 687 amino acid protein. It is expressed as a 3.6 kb mRNAin human endothelial cells. Tissue transglutaminase 3 (TG3) has theGenbank accession number L10386, and encodes a 692 amino acid protein.It is expressed as a 2.9-kb mRNA. The sequences of TG2 and TG3 find usein the recombinant production of the encoded polypeptide.

Transglutaminase polypeptides can be produced through isolation fromnatural sources, recombinant methods and chemical synthesis. Inaddition, functionally equivalent polypeptides may find use, where theequivalent polypeptide may contain deletions, additions or substitutionsof amino acid residues that result in a silent change, thus producing afunctionally equivalent differentially expressed on pathway geneproduct. Amino acid substitutions may be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity, and/orthe amphipathic nature of the residues involved. “Functionallyequivalent”, as used herein, refers to a protein capable of exhibiting asubstantially similar activity as the native polypeptide.

The polypeptides may be produced by recombinant DNA technology usingtechniques well known in the art. Methods that are well known to thoseskilled in the art can be used to construct expression vectorscontaining coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized.

As described in the examples, during normal digestion, a peptidaseresistant oligopeptide core remains after exposure of glutens, e.g.gliadin, to normal digestive enzymes. Oligopeptide fragments of interestinclude fragments of at least about 20 contiguous amino acids, moreusually at least about 33 contiguous amino acids, and may comprise 50 ormore amino acids, and may extend further to comprise additionalsequences. Examples of other peptidase resistant oligopeptides areprovided in SEQ ID NO:5, 6, 7 and 10. Other examples of immunogenicgliadin oligopeptides are discussed by Wieser (1995) Baillieres ClinGastroenterol 9(2):191-207.

The multivalent gluten oligopeptides may be substituted with a glutamineanalog at one or more positions, e.g. to enhance formation of a complexor covalent binding between tTGase and the peptid analog. Analogs usefulin the preparation of substituted peptide for this purpose include thefollowing:

where R1 and R2 are independently selected from H, alkyl, alkenyl,cycloalkyl, aryl, heteroalkyl, heteroaryl, alkoxy, alkylthio, arakyl,aralkenyl, halo, haloalkyl, haloalkoxy, heterocyclyl, andheterocyclylalkyl groups. R1 and R2 may also comprise peptidicprotecting groups. The amino acid analogs, 6-diazo-5-oxo-norleucine(Don), Azaserine (Aza), 6-thio(tetramethyl imidazoyl)-5-oxo-norleucine(Ton), 2-[2-thio(tetramethyl imidazoyl)-acyl]-2,3-diaminopropionic acid(Tad), acivicin (Aci)) and 3-chloro-4,5-dihydro-5-amino-isoxazole arealso proposed as glutamine mimetics.

Polypeptide and Oligopeptide Compositions

The oligopeptides and proteins useful in the methods of the presentinvention may be prepared in accordance with conventional techniques,such as synthesis, recombinant techniques, isolation from naturalsources, or the like. For example, solid-phase peptide synthesisinvolves the successive addition of amino acids to create a linearpeptide chain (see Merrifield (1963) J. Am. Chem. Soc. 85:2149-2154).Production of a peptide or protein by recombinant DNA technology canalso be performed. Thus, the oligopeptides may be prepared by in vitrosynthesis, using conventional methods as known in the art. Variouscommercial synthetic apparatuses are available, for example, automatedsynthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman,and other manufacturers. By using synthesizers, naturally occurringamino acids may be substituted with unnatural amino acids. Theparticular sequence and the manner of preparation will be determined byconvenience, economics, purity required, and the like.

The sequence of the provided epitopes, and of amino acids flankingepitopes, may be altered in various ways known in the art to generatetargeted changes in sequence. Such “conservatively modified variants”will typically be functionally-preserved variants, which differ, usuallyin sequence, from the corresponding native or parent oligopeptide butstill retain the biological activity, i.e., epitopic specificity.Variants may also include fragments of the oligopeptide that retainactivity. Various methods known in the art may be used to generatetargeted changes, e.g. phage display in combination with random andtargeted mutations, introduction of scanning mutations, and the like.

A variant may be substantially similar to a native sequence, i.e.dffering by at least one amino acid, and may differ by at least two butnot more than about ten amino acids. The sequence changes may besubstitutions, insertions or deletions. Scanning mutations thatsystematically introduce alanine, or other residues, may be used todetermine key amino acids. Conservative amino acid substitutionstypically include substitutions within the following groups: (glycine,alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid);(asparagine, glutamine); (serine, threonine); (lysine, arginine); or(phenylalanine, tyrosine).

Modifications of interest that do not alter primary sequence includechemical derivatization of proteins, e.g., acetylation, orcarboxylation. Also included in the subject invention are oligopeptidesthat have been modified using molecular biological techniques andsynthetic chemistry so as to improve their resistance to proteolyticdegradation, to acidic conditions such as those found in the stomach, orto optimize solubility properties or to render them more suitable as atherapeutic agent. For examples, the backbone of the peptidase may becyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Analogs of such proteins include those containingresidues other than naturally occurring L-amino acids, e.g. D-aminoacids or non-naturally occurring synthetic amino acids. If desired,various groups may be introduced into the oligopeptide during synthesisor during expression, which allow for linking to other molecules or to asurface. Thus cysteines can be used to make thioethers, histidines forlinking to a metal ion complex, carboxyl groups for forming amides oresters, amino groups for forming amides, and the like.

Thus, the present invention includes oligopeptide analogs of theoligopeptides described by amino acid sequence herein. Such analogscontain at least one difference in amino acid sequence between theanalog and native antigenic peptide. An L-amino acid from the nativepeptide may be altered to any other one of the 20 L-amino acids commonlyfound in proteins, any one of the corresponding D-amino acids, rareamino acids, such as 4-hydroxyproline, and hydroxylysine, or anon-protein amino acid, such as β-alanine and homoserine. Also includedwith the scope of the present invention are amino acids that have beenaltered by chemical means such as methylation (e.g., α-methylvaline),deamidation, amidation of the C-terminal amino acid by an alkylaminesuch as ethylamine, ethanolamine, and ethylene diamine, and acylation ormethylation of an amino acid side chain function (e.g., acylation of theepsilon amino group of lysine), deimination of arginine to citrulline,isoaspartylation, or phosphorylation on serine, threonine, tyrosine orhistidine residues. Candidate oligopeptide analogs may be screened forutility in a diagnostic method of the invention by an assay m asuringcompetitive binding to MHC, and an assay measuring T cell proliferation.Those analogs that inhibit binding of the native peptides and thatstimulate proliferation of auto-reactive T cells are useful diagnosticreagents.

Oligopeptides and oligopeptide analogs may be synthesized by standardchemistry techniques, including synthesis by automated procedure. Ingeneral, peptide and peptide analogs are prepared by solid-phase peptidesynthesis methodology which involves coupling each protected amino acidresidue to a resin support, preferably a 4-methylbenzhydrylamine resin,by activation with dicyclohexylcarbodiimide to yield a peptide with aC-terminal amide. Alternatively, a chloromethyl resin (Merrifield resin)may be used to yield a peptide with a free carboxylic acid at theC-terminus. After the last residue has been attached, the protectedpeptide-resin is treated with hydrogen fluoride to cleave the peptidefrom the resin, as well as deprotect the side chain functional groups.Crude product can be further purified by gel filtration, HPLC, partitionchromatography, or ion-exchange chromatography.

The oligopeptides may also be isolated and purified in accordance withconventional methods of recombinant synthesis, or from natural sources.A lysate may be prepared of the expression host and the lysate purifiedusing HPLC, exclusion chromatography, gel electrophoresis, affinitychromatography, or other purification technique. For the most part, thecompositions which are used will comprise at least 20% by weight of thedesired product, more usually at least about 75% by weight, preferablyat least about 95% by weight, and for diagnostic purposes, usually atleast about 99.5% by weight, in relation to contaminants related to themethod of preparation of the product and its purification. Usually, thepercentages will be based upon total protein.

The peptides and proteins of the invention can also be used to generateother useful reagents of the invention, including monoclonal andpolyclonal antibody-producing cell lines and antibodies derivedtherefrom in isolated and purified form. The peptides and proteins ofthe invention can also be used to generate highly purified preparationsof T cell lines, the proliferation of which is stimulated by thosepeptides and proteins. Such cell lines and antibodies are useful indiagnostic methods of the invention.

Diagnostic Methods

The present invention provides a variety of methods for diagnosingCeliac Sprue. In one embodiment, the diagnosis involves detecting thepresence of a gluten oligopeptides digestion product, e.g. SEQ ID NO:12;deamidated counterparts there; a tTGase-linked counterpart thereof;etc., in a tissue, bodily fluid, or stool of an individual. Thedetecting step can be accomplished by use of a reagent, e.g. anantibody, that recognizes the indicated antigen, or by a cell thatproliferates in the presence of the indicated antigen and suitableantigen presenting cells, wherein said antigen presenting cells arecompatible with the MHC type of the proliferating cell, e.g. allogeneiccells, autologous cells, etc.

In another embodiment, the diagnosis involves detecting the presence ofan antibody, one or more T cells reactive with the 33-mer or adeamidated counterpart thereof, or a tTGase-linked counterpart thereofin a tissue, bodily fluid, or stool of an individual. In one embodiment,an antibody is detected by, for example, an agglutination assay using anantigen provided by the present invention. In another embodiment, a Tcell is detected by its proliferation in response to exposure to amultivalent gluten oligopeptide provided by the present invention andpresented by autologous or suitable allogeneic antigen presenting cells.

In one aspect, the methods and reagents of the present invention arecapable of detecting the toxic oligopeptides of gluten proteins ofwheat, barley, oats and rye remaining after digestion or partialdigestion of the same by a Celiac Sprue individual. Gluten is theprotein fraction in cereal dough, which can be subdivided into gluteninsand prolamines, which can be further subclassified as gliadins,secalins, hordeins, avenins from wheat, rye, barley and oat,respectively. For further discussion of gluten proteins, see the reviewby Wieser (1996) Acta Paediatr Suppl. 412:3-9; herein incorporated byreference. Among gluten proteins of interest are included the storageproteins of wheat, species of which include Triticum aestivum; Triticumaethiopicum; Triticum baeoticum; Triticum militinae; Triticummonococcum; Triticum sinskajae; Triticum timopheevii; Tnticum turgidum;Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; and the like.A review of the genes encoding wheat storage proteins may be found inColot (1990) Genet Eng (N Y) 12:22541.

Of particular interest is gliadin, which is the alcohol-soluble proteinfraction of wheat gluten. Gliadins are typically rich in glutamine andproline, particularly in the N-terminal part. For example, the first 100amino acids of α- and γ-gliadins contain ˜35% and ˜20% of glutamine andproline residues, respectively. Many wheat gliadins have beencharacterized, and as there are many strains of wheat and other cereals,it is anticipated that many more sequences will be obtained usingroutine methods of molecular biology. Examples of sequenced gliadinsinclude wheat alpha gliadin sequences, for example as provided inGenbank, accession numbers AJ133612; AJ133611; AJ133610; AJ133609;AJ133608; AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602;D84341.1; U51307; U51306; U51304; U51303; U50984; and U08287. A sequenceof wheat omega gliadin is set forth in Genbank accession numberAF280605.

For the purposes of the present invention, toxic gliadin oligopeptidesare peptides derived during normal digestion of gliadins and relatedstorage proteins as described above, from dietary cereals, e.g. wheat,rye, barley, and the like, by a Celiac Sprue individual. Sucholigopeptides are believed to act as antigens for T cells in CeliacSprue individuals. For binding to Class II MHC proteins, immunogenicpeptides are usually from about 6 to 20 amino acids in length, moreusually from about 10 to 18 amino acids, and as demonstrated herein, aparticularly stimulatory toxic gliadin oligopeptide is the multivalent33-mer d scribed above. Such peptides include PXP motifs, for examplethe motif PQPQLP (SEQ ID NO:8). Determination of whether an oligopeptideis immunogenic for a particular patient is r adily determined bystandard T cell activation assays known to those of skill in the art.Illustrative toxic gliadin oligopeptides of the invention are describedin Examples I and 2 below. The 33-mer gliadin oligopeptide of Example 2and its deamidated counterpart formed by tTGase are preferred toxicgliadin oligopeptides of the invention.

Samples may be obtained from patient tissue, which may be a mucosaltissue, including but not limited to oral, nasal, lung, and intestinalmucosal tissue, a bodily fluid, e.g. blood, sputum, urine, phlegm,lymph, and tears. One advantage of the present invention is that theantigens provided are such potent antigens, both for antibody-bindingand T-cell stimulation, that the diagnostic methods of the invention canbe employed with samples (tissue, bodily fluid, or stool) in which aCeliac Sprue diagnostic antibody, peptide, or T cell is present in verylow abundance. This allows the methods of the invention to be practicedin ways that are much less invasive, much less expensive, and much lessharmful to the Celiac Sprue individual.

Patients may be monitored for the presence of reactive T cells, usingone or more multivalent oligopeptides as described above. The presenceof such reactive T cells indicates the presence of an on-going immuneresponse. The antigen used in the assays is a multivalent glutenoligopeptide as described above; including, without limitation, SEQ IDNO:12; deamidated counterparts; tTGase fusions thereof; and derivatives.Cocktails comprising multiple oligopeptides; panels of peptides; etc.may be also used. Overlapping peptides may be generated, where eachpeptide is frameshifted from 1 to 5 amino acids, thereby generating aset of epitopes.

The diagnosis may determine the level of reactivity, e.g. based on thenumber of reactive T cells found in a sample, as compared to a negativecontrol from a naive host, or standardized to a data curve obtained fromone or more positive controls. In addition to detecting the qualitativeand quantitative presence of antigen reactive T cells, the T cells maybe typed as to the expression of cytokines known to increase or suppressinflammatory responses. While not necessary for diagnostic purposes, itmay also be desirable to type the epitopic specificity of the reactive Tcells, particularly for use in therapeutic administration of peptides.

T cells may be isolated from patient peripheral blood, lymph nodes,including peyer's patches and other gut-related lymph nodes, or fromtissue samples as described above. Reactivity assays may be performed onprimary T cells, or the cells may be fused to generate hybridomas. Suchreactive T cells may also be used for further analysis of diseaseprogression, by monitoring their in situ location, T cell receptorutilization, MHC cross-reactivity, etc. Assays for monitoring T cellresponsiveness are known in the art, and include proliferation assaysand cytokine release assays. Also of interest is an ELISA spot assay.

Proliferation assays measure the level of T cell proliferation inresponse to a specific antigen, and are widely used in the art. In onesuch assay, recipient lymph node, blood or spleen cells are obtained atone or more time points after transplantation. A suspension of fromabout 10⁴ to 10⁷ cells, usually from about 10⁵ to 10⁶ cells is preparedand washed, then cultured in the presence of a control antigen, and testantigens, as described above. The cells are usually cultured for severaldays. Antigen-induced proliferation is assessed by the monitoring thesynthesis of DNA by the cultures, e.g. incorporation of ³H-thymidineduring the last 18 H of culture.

T cell cytotoxic assays measure the numbers of cytotoxic T cells havingspecificity for the test antigen. Lymphocytes are obtained at differenttime points after transplantation. Alloreactive cytotoxic T cells aretested for their ability to kill target cells bearing recipient MHCclass I molecules associated with peptides derived from a test antigen.In an exemplary assay, target cells presenting peptides from the testantigen, or a control antigen, are labeled with Na⁵¹CrO₄. The targetcells are then added to a suspension of candidate reactive lymphocytes.The cytotoxicity is measured by quantitating the release of Na⁵¹CrO₄from lysed cells. Controls for spontaneous and total release aretypically included in the assay. Percent specific ⁵¹Cr release may becalculated as follows: 100×(release by CTL−spontaneous release)/(totalrelease−spontaneous release).

Enzyme linked immunosorbent assay (ELISA) and ELISA spot assays are usedto determine the cytokine profile of reactive T cells, and may be usedto monitor for the expression of such cytokines as IL-2, IL4, IL-5,γIFN, etc. The capture antibodies may be any antibody specific for acytokine of interest, where supernatants from the T cell proliferationassays, as described above, are conveniently used as a source ofantigen. After blocking and washing, labeled detector antibodies areadded, and the- concentrations of protein present determined as afunction of the label that is bound.

In one embodiment of the invention, the presence of reactive T cells isdetermined by injecting a dose of the 33-mer peptide, or a derivative orfragment thereof as described above, subcutaneously or sub-mucosallyinto a patient, for example into the oral mucosa (see Lahteenoja et al.(2000) Am. J. Gastroenterology 95:2880, herein incorporated byreference). A control comprising medium alone, or an unrelated proteinis usually injected nearby at the same time. The site of injection isexamined after a period of time, by biopsy or for the presence of awheal.

A wheal at the site of injection is compared to that at the site of thecontrol injection, usually by measuring the size of the wheal. The skintest readings may be assessed by a variety of objective grading systems.A positive result for the presence of an immune response will show anincreased diameter at the site of polypeptide injection as compared tothe control.

Where a biopsy is performed, the specimen is examined for the presenceof increased numbers of immunologically active cells, e.g. T cells, Bcells, mast cells, and the like. For example, methods of histochemistryand/or immunohistochemistry may be used, as is known in the art. Thedensities of cells, including antigen specific T cells, mast cells, Bcells, etc. may be examined. It has been reported that increased numbersof intraepithelial CD8+T cells may correlate with gliadin reactivity.

An alternative method relies on the detection of circulating antibodiesin a patient. Methods of detecting specific antibodies are well-known inthe art. Antibodies specific for multivalent gluten oligopeptides asdescribed above may be used in screening immunoassays. A sample is takenfrom the patient. Samples, as used herein, include biological fluidssuch as blood, tears, saliva, lymph, dialysis fluid and the like; organor tissue culture derived fluids; and fluids extracted fromphysiological tissues. Also included in the term are derivatives andfractions of such fluids. Blood samples and derivatives thereof are ofparticular interest.

Measuring the concentration of specific antibodies in a sample orfraction thereof may be accomplished by a variety of specific assays. Ingeneral, the assay will measure the reactivity between a patient sample,usually blood derived, generally in the form of plasma or serum. Thepatient sample may be used directly, or diluted as appropriate, usuallyabout 1:10 and usually not more than about 1:10,000. Immunoassays may beperformed in any physiological buffer, e.g. PBS, normal saline, HBSS,dPBS, etc.

In one embodiment, a conventional sandwich type assay is used. Asandwich assay is performed by first attaching the peptide to aninsoluble surface or support. The peptide may be bound to the surface byany convenient means, depending upon the nature of the surface, eitherdirectly or through specific antibodies. The particular manner ofbinding is not crucial so iong as it is compatible with the reagents andoverall methods of the invention. They may be bound to the platescovalently or non-covalently, preferably non-covalently.

The insoluble supports may be any composition to which peptides can bebound, which is readily separated from soluble material, and which isotherwise compatible with the overail method of measuring antibodies.The surface of such supports may be solid or porous and of anyconvenient shape. Examples of suitable insoluble supports to which thereceptor is bound include beads, e.g. magnetic beads, membranes andmicrotiter plates. These are typically made of glass, plastic (e.g.polystyrene), polysaccharides, nylon or nitrocellulose. Microtiterplates are especially convenient because a large number of assays can becarried out simultaneously, using small amounts of reagents and samples.

Before adding patient samples or fractions thereof, the non-specificbinding sites on the insoluble support i.e. those not occupied byantigen, are generally blocked. Preferred blocking agents includenon-interfering proteins such as bovine serum albumin, casein, gelatin,and the like. Alternatively, several detergents at non-interf ringconcentrations, such as Tween, NP40, TX100, and the like may be used.

Samples, fractions or aliquots thereof are then added to separatelyassayable supports (for example, separate wells of a microtiter plate)containing support-bound antigenic peptide. Preferably, a series ofstandards, containing known concentrations of antibodies is assayed inparallel with the samples or aliquots thereof to serve as controls.

Generally from about 0.001 to 1 ml of sample, diluted or otherwise, issufficient, usually about 0.01 ml sufficing. Preferably, each sample andstandard will be added to multiple wells so that mean values can beobtained for each. The incubation time should be sufficient forantibodies molecules to bind the insoluble antigenic peptide. Generally,from about 0.1 to 3 hr is sufficient, usually 1 hr sufficing.

After incubation, the insoluble support is generally washed of non-boundcomponents. Generally, a dilute non-ionic detergent medium at anappropriate pH, generally 7-8, is used as a wash medium. From one to sixwashes may be employed, with sufficient volume to thoroughly washnon-specifically bound proteins present in the sample.

After washing, a solution containing a second receptor specific for thepatient antibodies is applied. The receptor may be any compound thatbinds patient antibodies with sufficient specificity such that it can bedistinguished from other components present. In a preferred embodiment,second receptors are antibodies specific for patient antibodies, eithermonoclonal or polyclonal sera, e.g. mouse anti-human antibodies, mouseanti-dog antibodies, rabbit anti-cat antibodies, etc. Such second stageantibodies may be labeled to facilitate direct, or indirectquantification of binding. Examples of labels which permit directmeasurement of second receptor binding include radiolabels, such as ³Hor ¹²⁵I, fluorescers, dyes, beads, chemilumninescers, colloidalparticles, and the like. Examples of labels that permit indirectmeasurement of binding include enzymes where the substrate may providefor a colored or fluorescent product. In a preferred embodiment, thesecond receptors are antibodies labeled with a covalently bound enzymecapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. Alternatively, the second stage may beunlabeled, and a labeled third stage is used. Examples of secondreceptor/second receptor-specific molecule pairs includeantibodylanti-antibody and avidin (or streptavidin)lbiotin. Since theresultant signal is thus amplified, this technique may be advantageouswhere only a small amount of antibodies is present.

After the second stage has bound, the insoluble support is generallyagain washed free of non-specifically bound molecules, and the signalproduced by the bound conjugate is detected by conventional means. Wherean enzyme conjugate is used, an appropriate enzyme substrate is providedso a detectable product is formed. More specifically, where a peroxidaseis the selected enzyme conjugate, a preferred substrate combination isH₂O₂ and is O-phenylenediamine, which yields a colored product underappropriate reaction conditions. Appropriate substrates for other enzymeconjugates such as those disclosed above are known to those skilled inthe art. Suitable reaction conditions as well as means for detecting thevarious useful conjugates or their products are also known to thoseskilled in the art. For the product of the substrate O-phenylenediaminefor example, light absorbance at 490495 nm is conveniently measured witha spectrophotometer.

Generally the amount of bound antibodies detected will be compared tocontrol samples from normal patients. The presence of increased levelsof the antigen specific antibodies is indicative of disease, usually atleast about a 5 fold, 10 fold, or 100 fold increase will be taken as apositive reaction.

In some cases, a competitive assay will be used. In addition to thepatient sample, a competitor to the antibodies is added to the reactionmix. The competitor and the antibodies compete for binding to theantigenic peptide. Usually, the competitor molecule will be labeled anddetected as previously described, where the amount of competitor bindingwill be proportional to the amount of antibodies present. Theconcentration of competitor molecule will be from about 10 times themaximum anticipated antibodies concentration to about equalconcentration in order to make the most sensitive and linear range ofdetection.

An alternative protocol is to provide anti-patient antibodies bound tothe insoluble surface. After adding the sample and washing awaynon-specifically bound proteins, one or a combination of the testantigens are added, where the antigens are labeled, so as not tointerfere with binding to the antibodies. Conveniently, fused proteinsmay be employed, where the peptide sequence is fused to an enzymesequence, e.g. β-galactosidase.

It is particularly convenient in a clinical setting to perform theimmunoassay in a self-contained apparatus. A number of such methods areknown in the art. The apparatus will generally employ a continuousflow-path of a suitable filter or membrane, having at least threeregions, a fluid transport region, a sample region, and a measuringregion. The sample region is prevented from fluid transfer contact withthe other portions of the flow path prior to receiving the sample. Afterthe sample region receives the sample, it is brought into fluid transferrelationship with the other regions, and the fluid transfer regioncontacted with fluid to permit a reagent solution to pass through thesample region and into the measuring region. The measuring region mayhave bound to it the antigenic peptide, with a conjugate of an enzymewith an antibodies specific antibody employed as a reagent, generallyadded to the sample before application. Alternatively, the antigenicpeptide may be conjugated to an enzyme, with antibodies specificantibody bound to the measurement region.

Thus, in one aspect, the present invention provides a method fordiagnosing Celiac Sprue in an individual who has not consumed gluten foran extended period of time, such time including but not limited to oneday, one week, one month, and one year prior to the performance of thediagnostic method. The advantage conferred by this aspect of theinvention is that current diagnosis of a Celiac Sprue individualtypically involves a preliminary diagnosis, after which the individualis placed on a gluten-free diet. If the individual's symptoms abateafter initiation of the gluten-free diet, then the individual ischallenged with gluten, and another diagnostic test, such as anendoscopy or T cell proliferation assay, is performed to confirm thepreliminary diagnosis. This re-challenge with gluten causes extremediscomfort to the Celiac Sprue individual. One important benefitprovided by certain embodiments of the invention is that such are-challenge need not be performed to diagnose Celiac Sprue, becauseeven very low levels of 33-mer specific antibodies and T cell responderscan be identified using the methods of the invention.

In another aspect, the present invention provides a method fordiagnosing Celiac Sprue by detecting the presence of a 33-mer specificantibody or a T cell responder in a bodily tissue or fluid other thanintestinal mucosa. In this aspect of the invention, the diagnosticmethods are performed without recourse to endoscopy or intestinalbiopsy, thus avoiding the discomfort, pain, and expense attendant onsuch procedures in common use today.

The subject methods are useful not only for diagnosing Celiac Sprueindividuals but also for determining the efficacy of prophylactic ortherapeutic methods for Celiac Sprue as well as the efficacy of foodpreparation or treatment methods aimed at removing glutens or similarsubstances from food sources. Thus, a Celiac Sprue individualefficaciously treated with a prophylactic or therapeutic drug or othertherapy for Celiac Sprue tests more like a non-Celiac Sprue individualwith the methods of the invention. Likewise, the antibodies or T cellresponders, e.g. T cell lines, of the invention that detect the toxicgluten oligopeptides of the invention are useful in detecting gluten andgluten-like substances in food and so can be used to determine whether afood treated to remove such substances has been efficaciously treated.

As used herein, the term “treating” is used to refer to both preventionof disease, and treatment of pre-existing conditions. The treatment ofongoing disease, to stabilize or improve the clinical symptoms of thepatient, is of particular interest. Such treatment is desirablyperformed prior to loss of function in the affected tissues. Evidence oftherapeutic effect may be any diminution in the severity of disease,particularly measuring the severity of such symptoms as fatigue, chronicdiarrhea, malabsorption of nutrients, weight loss, abdominal distension,and anemia. Other disease indicia include the presence of antibodiesspecific for the 33-mer of the invention or its deamidated counterparts,glutens, antibodies specific for tissue transglutaminase or tTGase linkd to the 33-mer of the invention or its deamidated counterparts, thepresence of pro-inflammatory T cells and cytokines, histologicalexamination of the villus structure of the small intestine, and thelike. Patients may be adult or child, where children in particularbenefit from prophylactic treatment, as prevention of early exposure totoxic gluten peptides may prevent initial development of the disease.Children suitable for prophylaxis can be identified by genetic testingfor predisposition, e.g. by HLA typing; by family history, and,preferably, by the diagnostic methods of the present invention.

Although the present invention is not to be bound by any theory ofaction of the glutenases, it is believed that the primary event inCeliac Sprue requires intact gluten oligopeptides such as the 33-mer ofthe invention to gain access to antigen binding sites within the laminapropria region interior to the relatively impermeable surface intestinalepithelial layer. Ordinarily, oligopeptide end products of pancreaticprotease processing are rapidly and efficiently hydrolyzed into aminoacids, di- or tri-peptides by gastric peptidases before they can betransported across the epithelial layer. However, glutens have beenfound to be particularly peptidase resistant, which may be attributed tothe usually high proline content of these proteins, a residue that isinaccessible to most gastric peptidases.

The normal assimilation of dietary proteins by the human gut can bedissected into three major phases: (i) initiation of proteolysis in thestomach by pepsin and highly efficient endo- and C-terminal cleavage inthe upper small intestine cavity (duodenum) by secreted pancreaticproteases and carboxypeptidases; (ii) further processing of theresulting oligopeptide fragments by exo- and endopeptidases anchored inthe brush border surface membrane of the upper small intestinalepithelium aejunum); and (iii) facilitated transport of the resultingamino acids, di- and tripeptides across the epithelial cells into thelamina propria, from where these nutrients enter capillaries fordistribution throughout the body. Because most proteases and peptidasesare unable to hydrolyze the amide bonds of proline residues, it is shownherein that the abundance of proline residues in gliadins and relatedproteins from wheat, rye and barley can constitute a major digestiveobstacle for the enzymes involved in phases (i) and (ii) above. Thisleads to an increased concentration of relatively stable gluten derivedoligopeptides in the gut.

Tissue transglutaminase (tTGase), an enzyme found on the extracellularsurface in many organs including the intestine, catalyzes the formationof isopeptide bonds between glutamine and lysine residues of differentpolypeptides, leading to protein-protein crosslinks in the extracellularmatrix. The tTGase enzyme is the primary focus of the autoantibodyresponse in Celiac Sprue. Gliadins, secalins and hordeins containseveral sequences rich in Pro-Gin residues that are high-affinitysubstrates for tTGase; tTGase catalyzed deamidation of at least some ofthese sequences, such as, in particular, the 33-mer oligopeptide of theinvention, dramatically increases their affinity for HLA-DQ2, the class11 MHC allele present in >90% Celiac Sprue patients; and presentation ofthese deamidat d epitopes by DQ2 positive antigen presenting cellseffectively stimulates proliferation of gliadin-specific T cells fromintestinal biopsies of most Celiac Sprue patients. Proposed toxiceffects of gluten include immunogenicity of the gluten oligopeptides,leading to inflammation, including by a mechanism in which gliadinpeptides directly bind to surface receptors.

The various methods and reagents of the invention can be prepared andmodified as described below. Although specific methods and reagents areexemplified in the discussion below, it is understood that any of anumber of alternative methods, including those described above areequally applicable and suitable for use in practicing the invention. Itwill also be understood that an evaluation of the methods of theinvention may be carried out using procedures standard in the art,including the diagnostic and assessment methods described above.

The practice of the present invention may employ conventional techniquesof molecular biology (including recombinant techniques), microbiology,cell biology, biochemistry and immunology, which are within the scope ofthose of skill in the art. Such techniques are explained fully in theliterature, such as, “Molecular Cloning: A Laboratory Manual”, secondedition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J.Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987);“Methods in Enzymology” (Academic Press, Inc.); “Handbook ofExperimental Immunology” (D. M. Weir & C. C. Blackwell, eds.); “GeneTransfer Vectors for Mammalian Cells” (J. M. Miller & M. P. Calos, eds.,1987); “Current Protocols in Molecular Biology” (F. M. Ausubel et al.,eds., 1987); “PCR: The Polymerase Chain Reaction” (Mullis et al., eds.,1994); and “Current Protocols in Immunology” (J. E. Coligan et al.,eds., 1991).

As noted above, the subject methods are useful to monitor the progressand efficacy of therapies to treat individuals suffering from CeliacSprue and/or dermatitis herpetiformis. Such therapies can involveadministration of an effective dose of glutenase and/or tTGaseinhibitor, through a pharmaceutical formulation, incorporating glutenaseinto food products, administering live organisms that express glutenase,and the like. As these therapies may not have been approved by the FDAor an equivalent other regulatory agency, the methods of the inventionhave application in clinical trials conducted to evaluate the safety andefficacy of such therapies. Diagnosis of suitable patients may utilize avariety of criteria known to those of skill in the art in addition tothose methods described herein. A quantitative increase in antibodiesspecific for gliadin, and/or tissue transglutaminase is indicative ofthe disease.

Family histories and the presence of the HLA alleles HLA-DQ2[DQ(a1*0501, b1*02)] and/or DQ8 [DQ(a1*0301, b1*0302)] are indicative ofa susceptibility to the disease.

In addition to employing the diagnostic methods of the invention, thetherapeutic effect may be measured in terms of clinical outcome, or mayrely on immunological or biochemical tests. Suppression of thedeleterious T-cell activity can be measured by enumeration of reactiveTh1 cells, by quantitating the release of cytokines at the sites oflesions, or using other assays for the presence of autoimmune T cellsknown in the art. Alternatively, one may look for a reduction insymptoms of a disease.

Related applications include U.S. Provisional application No. 60/357,238filed Feb. 14, 2002; to U.S. Provisional Application No. 60/380,761filed May 14, 2002; to U.S. Provisional Application No. 60/392,782 filedJun. 28, 2002; and U.S. Provisional application No. 60/422,933, filedOct. 31, 2002, each of which are herein specifically incorporated byreference.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLE 1 Immunodominant Peptides of Gliadin are Protease Resistant

Recent studies have identified a small number of immunodominant peptidesfrom gliadin, which account for most of the stimulatory activity ofdietary gluten on intestinal and peripheral T lymphocytes found inCeliac patients. The proteolytic kinetics of these immunodominantpeptides were analyzed at the small intestinal surface. Using brushborder membrane vesicles from adult rat intestines, it was shown thatthese proline-glutamine-rich peptides are exceptionally resistant toenzymatic processing, and that dipeptidyl peptidase IV and dipeptidylcarboxypeptidase are the rate-limiting enzymes in their digestion. Theseresults support the conclusions drawn from the tests described inExample 2 that incomplete digestion of gliadin, which results in theformation of the 33-mer oligopeptide and its deamidated counterpartformed by tTGase action, contributes to the disease symptoms of CeliacSprue and can be employed in improved diagnostic methods for CeliacSprue.

To dissect this complex process, liquid chromatography coupled massspectroscopy analysis (LC-MS-MS) was utilized to investigate thepathways and associated kinetics of hydrolysis of immunodominant gliadinpeptides treated with rat BBM preparations. Because the rodent is anexcellent small animal model for human intestinal structure andfunction, rat BBM was chosen as a suitable model system for thesestudies.

BBM fractions were prepared from rat small intestinal mucosa asdescribed by Ahnen et al. (1982) J. Biol. Chem. 257, 12129-35. Usingstandard assays, the specific activities of- the known BB peptidaseswere determined to be 127 μU/μg for Aminopeptidase N (APN, EC 3.4.11.2),60 μU/μg for dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), and 41 μU/μgfor dipeptidyl carboxypeptidase (DCP, EC 3.4.15.1). No prolineaminopeptidase (EC 3.4.11.5) or prolyl endopeptidase activity (PEP, EC3.4.21.26) activity was detectable (<5 μU/μg). Alkaline phosphatase andsucrase were used as control BBM enzymes with activities of 66 μU/μg and350 μU/μg, respectively.

BBM fractions were partially purified from the small intestinal mucosaof adult female rats maintained on an ad libitum diet of wheat-basedstandard rodent chow. Total protein content was determined by a modifiedmethod of Lowry with BSA as a standard. Alkaline phosphatase activitywas determined with nitrophenyl phosphate. Sucrase activity was measuredusing a coupled glucose assay. DPP IV, proline aminopeptidase and APNwere assayed continuously at 30° C. in 0.1 M Tris-HCl, pH 8.0,containing 1 mM of the p-nitroanilides (ε=8,800 M⁻¹ cm⁻¹) Gly-Pro-pNA,Pro-pNA or Leu-pNA, the latter in additional 1% DMSO to improvesolubility. DCP activity was measured in a 100 μl reaction as therelease of hippuric acid from Hip-His-Leu. PEP activity was determinedcontinuously with 0.4 mM Z-Gly-Pro-pNA in PBS:H₂O:dioxane (8:1.2:0.8) at30° C. One unit is defined as the consumption of 1 μmol substrate perminute.

DPP IV and DCP are both up-regulated by a high proline content in thediet. However, APN activity using standard substrates was found to behigher than DPP IV even when fed extreme proline rich diets. Also,although a higher DCP vs. CPP activity has been observed with the modelpeptide Z-GPLAP at saturating concentrations, a difference in Km valuescould easily account the reversed ratio measured in this study. 100 μMwas chosen as the initial peptide concentration, since non-saturatingkinetics (k_(cat)/K_(m)) were considered to be physiologically morerelevant than the maximal rates of hydrolysis (k_(cat)).

Proteolysis with the BBM preparation was investigated using the peptide(SEQ ID NO:1) QLQPFPQPQLPY, a product of chymotryptic digestion of α-9gliadin (Arentz-Hansen et al. (2000) J. Exp. Med. 191, 603-12). Thispeptide has been shown to stimulate proliferation of T cells isolatedfrom most Celiac Sprue patients, and hence is considered to possess animmunodominant epitope. It was subjected to BBM digestion, followed byLC-MS-MS analysis. A standard 50 μl digestion mixture contained 100 μMof synthetic peptide, 10 μM tryptophan and Cbz-tryptophan as internalstandards, and resuspended BBM preparations with a final prot in contentof 27 ng/μl and exogenous proteins, as indicated, in phosphate bufferedsaline. After incubation at 37° C. for the indicated time, the enzymeswere inactivated by heating to 95° C. for 3 minutes. The reactionmixtures were analyzed by LC-MS (SpectraSystem, ThermoFinnigan) using aC18 reversed phase column (Vydac 218TP5215, 2.1×150 mm) withwater:acetonitrile:formic acid (0.1%):trifluoroacetic acid (0.025%) asthe mobile phase (flow: 0.2 ml/min) and a gradient of 10% acetonitrilefor 3 minutes, 10-20% for 3 minutes, 20-25% for 21 minutes followed by a95% wash. Peptide fragments in the mass range of m/z=300-2000 weredetected by electrospray ionization mass spectroscopy using a LCQ iontrap, and their identities were confirmed by MSMS fragmentationpatterns.

While the parent peptide (SEQ ID NO:1) QLQPFPQPQLPY disappeared with anapparent half time of 35 min, several intermediates were observed toaccumulate over prolonged periods (FIG. 1A). The MS intensities(m/z=300-2000 g/mol) and UV₂₈₀ absorbances of the parent peptides (SEQID NO:1) QLQPFPQPQLPY and (SEQ ID NO:3) PQPQLPYPQPQLPY were found todepend linearly on concentration in the range of 6-100 μM. The referencepeptides (SEQ ID NO:4) PQPQLPYPQPQLP, (SEQ ID NO:5) QLQPFPQPQLP, (SEQ IDNO:6) QPQFPQPQLPY and (SEQ ID NO:7) QPFPQPQLP were generatedindividually by limited proteolysis of the parent peptides with 10 μg/mIcarboxypeptidase A (C-0261, Sigma) and/or 5.9 μg/ml leucineaminopeptidase (L-5006, Sigma) for 160 min. at 37° C. and analyzed byLC-MS as in FIG. 1.

Indeed, the subsequent processing of the peptide was substantiallyretarded (FIG. 1B). The identities of the major intermediates wereconfirmed by tandem MS, and suggested an unusually high degree ofstability of the (SEQ ID NO:8) PQPQLP sequence, a common motif in T cellstimulating peptides. Based on this data and the known amino acidpreferences of the BBM peptidases, the digestive breakdown of (SEQ IDNO:1) QLQPFPQPQLPY was reconstructed, as shown in the insert of FIG. 1B.The preferred pathway involves serial cleavage of the N-terminalglutamine and leucine residues by aminopeptidase N (APN), followed byremoval of the C-terminal tyrosine by carboxypeptidase P (CPP) andhydrolysis of the remaining N-terminal QP-dipeptide by DPP IV. As seenin FIG. 1B, the intermediate (SEQ ID NO:6) QPFPQPQLPY (formed by APNattack on the first two N-terminal residues) and its derivatives areincreasingly resistant to further hydrolysis. Because the high prolinecontent seemed to be a major cause for this proteolytic resistance,digestion was compared with a commercially available non-proline controlpeptide (SEQ ID NO:9) RRLIEDNEYTARG (Sigma, St. Louis, Mo.). Initialhydrolysis was much faster (t_(1/2)=10 min). More importantly, digestiveintermediates were only transiently observed and cleared completelywithin one hour, reflecting a continuing high specificity of the BBM forthe intermediate peptides.

Because the three major intermediate products (SEQ ID NO:6) QPFPQPQLPY,(SEQ ID NO:7) QPFPQPQLP, (SEQ ID NO:11) FPQPQLP) observed during BBMmediated digestion of (SEQ ID NO:1) QLQPFPQPQLPY are substrates for DPPIV, the experiment was repeated in the presence of a 6-fold excessactivity of exogenous fungal DPP IV. Whereas the relatively rapiddecrease of the parent peptide and the intermediate levels of (SEQ IDNO:5) QLQPFPQPQLP were largely unchanged, the accumulation of DPP IVsubstrates was entirely suppressed and complete digestion was observedwithin four hours. (FIG. 1B, open bars).

To investigate the rate-limiting steps in BBM mediated digestion ofgliadin peptides from the C-terminal end, another known immunodominantpeptide derived from wheat α-gliadin, (SEQ ID NO:3) PQPQLPYPQPQLPY, wasused. Although peptides with N-terminal proline residues are unlikely toform in the small intestine (none were observed during BBM digestion of(SEQ ID NO:1) QLQPFPQPQLPY, FIG. 1A), they serve as a useful model forthe analysis of C-terminal processing since the N-terminal end of thispeptide can be considered proteolytically inaccessible due to minimalproline aminopeptidase activity in the BBM. As shown in FIG. 2, thispeptide is even more stable than (SEQ ID NO:l) QLQPFPQPQLPY. Inparticular, removal of the C-terminal tyrosine residue bycarboxypeptidase P (CPP) is the first event in its breakdown, and morethan four times slower than APN activity on (SEQ ID NO:1) QLQPFPQPQLPY(FIG. 1B). The DCP substrate (SEQ ID NO:4) PQPQLPYPQPQLP emerges as amajor intermediate following carboxypeptidase P catalysis, and is highlyresistant to further digestion, presumably due to the low level ofendogenous DCP activity naturally associated with the BBM. To confirmthe role of DCP as a rate-limiting enzyme in the C-terminal processingof immunodominant gliadin peptides, the reaction mixtures weresupplemented with rabbit lung DCP. Exogenous DCP significantly reducedthe accumulation of (SEQ ID NO:4) PQPQLPYPQPQLP after overnightincubation in a dose dependent manner (FIG. 2C). Conversely, the amountof accumulated (SEQ ID NO:4) PQPQLPYPQPQLP increased more than 2-fold inthe presence of 10 μM of captopril, a DCP-specific inhibitor, ascompared with unsupplemented BBM.

Together, the above results demonstrate that (i) immunodominant gliadinpeptides are exceptionally stable toward breakdown catalyzed by BBMpeptidases, and (ii) DPP IV and especially DCP are rate-limiting stepsin this breakdown process at the N— and C-terminal ends of the peptides,respectively. Because BBM exopeptidases are restricted to N— or C—terminal processing, it was investigated if generation of additionalfree peptide ends by pancreatic enzymes would accelerate digestion. Ofthe pancreatic proteases tested, only elastase at a high(non-physiological) concentration of 100 ng/μl was capable ofhydrolyzing (SEQ ID NO:3) PQPQLPYPQPQ↓LPY. No proteolysis was detectedwith trypsin or chymotrypsin.

The above data demonstrates that proline-rich gliadin peptides areextraordinarily resistant to digestion by small intestinal endo- andexopeptidases, and therefore are likely to accumulate at highconcentrations in the intestinal cavity after a gluten rich meal. Thepathological implication of digestive resistance is strengthened by theobserved close correlation of proline content and celiac toxicity asobserved in the various common cereals (Schuppan (2000) Gastroenterology119, 234-42).

EXAMPLE 2 Immunodominant Peptide of Wheat Gliadin

It has long been known that the principal toxic components of wheatgluten are a family of closely related Pro-Gin rich proteins calledgliadins. Recent reports have suggested that peptides from a shortsegment of a-gliadin appear to account for most of the gluten-specificrecognition by CD4+ T cells from Celiac Sprue patients. These peptidesare substrates of tissue transglutaminase (tTGase), the primaryauto-antigen in Celiac Sprue, and the products of this enzymaticreaction bind to the class II HLA DQ2 molecule. This exampledemonstrates, using a combination of in vitro and in vivo animal andhuman studies, that this “immunodominant” region of α-gliadin is part ofan unusually long proteolytic product generated by the digestive processthat: (a) is exceptionally resistant to further breakdown by gastric,pancreatic and intestinal brush border proteases; (b) is the highestspecificity substrate of human tissue transglutaminase (tTGase)discovered to date; (c) contains at least six overlapping copies ofepitopes known to be recognized by patient derived T cells; (d)stimulates representative T cell clones that recognize these epitopeswith sub-micromolar efficacy; and (e) has homologs in proteins from alltoxic foodgrains but no homologs in non-toxic foodgrain proteins. Inaggregate, these findings demonstrate that the onset of symptoms upongluten exposure can be traced back to a small segment of a-gliadin.Finally, it is shown that this “super-antigenic” long peptide can bedetoxified in vitro and in vivo by treatment with bacterial prolylendopeptidase, providing a strategy for peptidase therapy for CeliacSprue.

Identification of stable peptides from gastric protease, pancreaticprotease and brush border membrane peptidase catalyzed digestion ofrecombinant α2-gliadin: α2-gliadin, a representative α-gliadin(Arentz-Hansen et al. (2000) Gut 46:46), was expressed in recombinantform and purified from E. coli. The α2-gliadin gene was cloned in pET28aplasmid (Novagen) and transformed into the expression host BL21(DE3)(Novagen). The transformed cells were grown in 1-liter cultures of LBmedia containing 50 μg/ml of kanamycin at 37° C. until the OD600 0.6-1was achieved. The expression of α2-gliadin protein was induced with theaddition of 0.4 mM isopropyl β-D-thiogalactoside (Sigma), and thecultures were further incubated at 37° C. for 20 hours. The cellsexpressing the recombinant α2-gliadin were centrifuged at 3600 rpm for30 minutes. The pellet was resuspended in 15 ml of disruption buffer(200 mM sodium phosphate; 200 mM NaCl; 2.5 mM DTT; 1.5 mM benzamidine;2.5 mM EDTA; 2 mg/L pepstatin; 2 mg/L leupeptin; 30% v/v glycerol) andlysed by sonication (1 minute; output control set to 6). Aftercentrifugation at 45000g for 45 min, the supematant was discarded andthe pellet containing gliadin protein was resuspended in 50 ml of 7Murea in 50 mM Tris (pH=8.0). The suspension was again centrifuged at45000g for 45 min and the supernatant was harvested for purification.

The supernatant containing α2-gliadin was incubated with I ml ofnickel-nitrilotriacetic acid resin (Ni-NTA; Qiagen) overnight and thenbatch-loaded on a column with 2 ml of Ni— NTA. The column was washedwith 7 M urea in 50 mM Tris (pH =8.0), and α2-gliadin was eluted with200 mM imidazole, 7 M urea in 50 mM Tris (pH=4.5). The fractionscontaining α2-gliadin were pooled into a final concentration of 70%ethanol solution, and two volumes of 1.5 M NaCl were added toprecipitate the protein. The solution was incubated at 4° C. overnight,and the final precipitate was collected by centrifugation at 45000 g for30 min., rinsed in water, and re-centrifuged to remove the urea. Thefinal purification step of the α-2 gliadin was developed withreverse-phase HPLC. The Ni-NTA purified protein fractions were pooled in7 M urea buffer and injected to a Vydac (Hesperia, CA) polystyrenereverse-phase column (i.d. 4.6 mm×25 cm) with the starting solvent (30%of solvent B: 1:1 HPLC-grade acetonitrile/isopropanol:0.1% TFA). SolventA was an aqueous solution with 0.1% TFA. The separation gradientextended from 30-100% of solvent B over 120 min. at a flow rate of 0.8ml/min. TABLE 2 Amount of Peptides Digested after 15 hours 33-merControl A Control B H1P0 <20% >90% >90% H2P0 <20% >61% >85% H3P0<20% >87% >95% H4P0 <20% >96% >95% H5P0 <20% >96% >95%

The purity of the recombinant gliadin was >95%, which allowed for facileidentification and assignment of proteolytic products by LC-MS/MS/UV.Although many previous studies utilized pepsin/trypsin treated gliadins,it was found that, among gastric and pancreatic proteases, chymotrypsinplayed a major role in the breakdown of α2-gliadin, resulting in manysmall peptides from the C-terminal half of the protein and a few longer(>8 residues) peptides from the N-terminal half, the most noteworthybeing a relatively large fragment, the 33-mer (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF (residues 57-89). This peptide was ofparticular interest for two reasons: (a) whereas most other relativelystable proteolytic fragments were cleaved to smaller fragments when thereaction times were extended, the 33-mer peptide remained intact despiteprolonged exposure to proteases; and (b) three distinct patient-specificT cell epitopes identified previously are present in this peptide,namely, PFPQPQLPY, PQPQLPYPQ (3 copies), and PYPQPQLPY (2 copies).

To establish the physiological relevance of this peptide, compositegastric/pancreatic enzymatic digestion of α2 gliadin was then examined.As expected, enzymatic digestion with pepsin (1:100 w/w ratio), trypsin(1:100), chymotrypsin (1:100), elastase (1:500) and carboxypeptidase (1:100) was quite efficient, leaving behind only a few peptides longer than9 residues (the minimum size for a peptide to show class 11 MHC mediatedantigenicity) (FIG. 4). In addition to the above-mentioned 33-mer, thepeptide WQIPEQSR was also identified, and was used as a control in manyof the following studies. The stability of the 33-mer peptide can alsobe appreciated, when comparing the results of a similar experiment usingmyoglobin (another common dietary protein). Under similar proteolyticconditions, myoglobin is rapidly broken down into much smaller products.No long intermediate is observed to accumulate.

The small intestinal brush-border membrane (BBM) enzymes are known to bevital for breaking down any remaining peptides from gastric/pancreaticdigestion into amino acids, dipeptides or tripeptides for nutritionaluptake. Therefore a comprehensive analysis of gliadin metabolism alsorequired investigations into BBM processing of gliadin peptides ofreasonable length derived from gastric and pancreatic proteasetreatment. BBM fractions were prepared from rat small intestinal mucosa.The specific activities of known BBM peptidases were verified to bewithin the previously reported range. Whereas the half-life ofdisappearance of WQIPEQSR was ˜60 min. in the presence of 12 ng/μl BBMprotein, the half-life of (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF digestion was >20 h. Therefore, thelatter peptide remains intact throughout the digestive process in thestomach and upper small intestine, and is poised to act as a potentialantigen for T cell proliferation and intestinal toxicity in geneticallysusceptible individuals.

Verification of proteolytic resistance of the 33-mer gliadin peptidewith brush border membrane preparations from human intestinal biopsies:To validate the above conclusions, derived from studies with rat BBMpreparations, in the context of human intestinal digestion, BBMpreparations were prepared from a panel of adult human volunteers, oneof whom was a Celiac Sprue patient in remission, while the rest werefound to have normal intestinal histology. (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, (SEQ ID NO:1) QLQPFPQPQLPY (aninternal sequence from the 33-mer used as a control), WQIPEQSR and othercontrol peptides (100 μM) were incubated with BBM prepared from eachhuman biopsy (final aminopeptidase N activity of 13 μU/μl) at 37° C. forvarying time periods. While QLQPFPQPQLPY, WQIPEQSR and other controlpeptides were completely proteolyzed within 1-5 h, the long peptideremained largely intact for at 19 hours. These results confirm theequivalence between the rat and human BBM for the purpose of this study.

Verification of proteolytic resistance of the 33-mergliadin peptide inintact animals: The proteolytic resistance of the 33-mer gliadinpeptide, observed in vitro using BBM from rats and humans, was confirmedin vivo using a perfusion protocol in intact adult rats (Smithson andGray (1977) J. Clin. Invest. 60:665). Purified peptide solutions wereperfused through a 15-20 cm segment of jejunum in a sedated rat with aresidence time of 20 min., and the products were collected and subjectedto LC-MS analysis. Whereas >90% of (SEQ ID NO:1) QLQPFPQPQLPY wasproteolyzed in the perfusion experiment, most of the 33-mer gliadinpeptide remained intact. These results demonstrate that the 33-merpeptide is very stable as it is transported through the mammalian uppersmall intestine.

The 33-mer gliadin peptide is an excellent substrate for tTGase, and theresulting product is a highly potent activator of patient-derived Tcells. A number of recent studies have demonstrated that regiospecificdeamidation of immunogenic gliadin peptides by tTGase increases theiraffinity for HLA-DQ2 as well as the potency with which they activatepatient-derived gluten-specific T cells. It has been shown thespecificity of tTGase for certain short antigenic peptides derived fromgliadin is higher than its specificity toward its physiological targetsite in fibronectin, for example, the specificity of tTGase for thea-gliadin derived peptide PQPQLPYPQPQLPY is 5-fold higher than that forits target peptide sequence in fibrinogen, its natural substrate. Thekinetics and regiospecificity of deamidation of the 33-mer α-gliadinpeptide identified as above were therefore measured. The k_(cat)/K_(M)was higher than that reported for any peptide studied thus far:kcat/KM=440 min-1 mM−1 for (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF compared to kcat/KM=82 min-1mM-1 forPQPQLPY and kcat / KM =350 min-1 mM-1 for PQPQLPYPQPQLPY.

Moreover, LC-MS-MS analysis revealed that (SEQ ID NO:12)LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF was selectively deamidated by tTGaseat the underlined residues. Since tTGase activity is associated with thebrush border membrane of intestinal enterocytes, it is likely thatdietary uptake of even small quantities of wheat gluten will lead to thebuild-up of sufficient quantities of this 33-mer gliadin peptide in theintestinal lumen so as to be recognized and processed by tTGase.

Structural characteristics of the 33-mer gliadin peptide and itsnaturally occurring homologs: Sequence alignment searches using BLASTPin all non-redundant protein databases revealed several homologs(E-value <0.001) of the 33-mer gliadin peptide.

Interestingly, foodgrain derived homologs were only found in gliadins(from wheat), hordeins (from barley) and secalins (from rye), all ofwhich have been proven to be toxic to Celiac patients. See FIG. 6.Nontoxic foodgrain proteins, such as avenins (in oats), rice and maize,do not contain homologous sequences to the 33-mer gliadin. In contrast,a BLASTP search with the entire α2-gliadin sequence identified foodgrainprotein homologs from both toxic and nontoxic proteins. Based onavailable information regarding the substrate specificities of gastric,pancreatic and BBM proteases and peptidases, it is predicted that,although most gluten homologs to the 33-mer gliadin peptide containedmultiple proteolytic sites and are therefore unlikely to be completelystable toward digestion, several sequences from wheat, rye and barleyare expected to be comparably resistant to gastric and intestinalproteolysis. The stable peptide homologs to the 33-mer α2-gliadinpeptide are QPQPFPPQLPYPQTQPFPPQQPYPQPQPQYPQPQ (from α1- andα6-gliadins); QQQPFPQQPIPQQPQPYPQQPQPYPQQPFPPQQPF (from B1 hordein);QPFPQPQQTFPQQPQLPFPQQPQQPFPQPQ (from γ-gliadin);QPFPQPQQPTPIQPQQPFPQRPQQPFPQPQ (from γ-secalin). These stable peptidesare all located at the N-terminal region of the corresponding proteins.The presence of proline residues after otherwise cleavable residues inthese peptides would contribute to their proteolytic stability.

The unique primary sequence of the 33-mer gliadin peptide also hadhomologs among a few non-gluten proteins. Among the strongest homologswere internal sequences from pertactin (a highly immunogenic proteinfrom Bordetella pertussis) and a mammalian inositol-polyphosphate5-phosphatase of unknown function. In both cases available informationsuggested that the homology could have biologically relevance. Forexample, the region of pertactin that is homologous to the 33-mergliadin peptide is known to be part of the immunodominant segment of theprotein. In the case of the homologous phosphatase, the correspondingpeptide region of the phosphatase is known to be responsible forvesicular trafficking of the phosphatase to the cytoplasmic Golgi. Inanalogy with the current picture of how gliadin peptides are presentedto HLA-DQ2 via a tTGase mediated pathway, these Pro-Gin-rich segments ofboth pertactin and the phosphatase are likely to be good tTGasesubstrates. To test this hypothesis, the corresponding peptides weresynthesized, and the selectivity of tTGase for these peptides wasmeasured. As predicted, both peptides were found to be good substratesof tTGase. The tTGase enzyme plays a central role in receptor mediatedendocytosis of several biologically important proteins. The biologicalactivities of both pertactin and the phosphatase may depend on tTGasemediated trafficking.

Secondary structural studies using circular dichroism spectroscopy onthe 33-mer gliadin peptide as well as its homologs from pertactin andthe inositol-polyphosphate 5-phosphatase demonstrate that these peptideshave strong type II polyproline helical character. In addition toreinforcing the proteolytic resistance of these peptides, the type IIpolyproline helical conformation is also likely to enhance theiraffinity for class 11 MHC proteins.

Although gluten proteins from foodgrains such as wheat, rye and barleyare central components of a nutritious diet, they can be extremely toxicfor patients suffering from Celiac Sprue. To elucidate the structuralbasis of gluten toxicity in Celiac Sprue, comprehensive proteolyticanalysis was performed on a representative recombinant gliadin underphysiologically relevant conditions. An unusually long andproteolytically stable peptide product was discovered, whosephysiological relevance was confirmed by studies involving brush bordermembrane proteins from rat and human intestines as well as intestinalperfusion assays in live rats. In aggregate, these data demonstrate thatthis peptide and its homologs found in other wheat, rye and barleyproteins are the “root cause” of the initial inflammatory response todietary wheat in Celiac Sprue patients in remission.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. Moreover, due to biological functionalequivalency considerations, changes can be made in protein structurewithout affecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

1. A purified gluten oligopeptide having the formula:E₁-X₁-E₂-X₂-E₃ . . . X_(n)-E_(y), wherein E₁, E₂ and E₃ areindependently selected epitopes, X₁ and X₂ are independently selectedfrom the group consisting of a peptide bond and at least one amino acid,n=0-5, and y=0-5.
 2. The oligopeptide of claim 1, wherein said epitopesare independently selected from the group consisting of PFPQPQLPY,PQPQLPYPQ, PQLPYPQPQ, PYPQPQLPY, PQPELPYPQ, PFPQPELPY, PQQSFPQQQ,PFPQQPQQPFP, PYPQPELPY, and conservatively modified variants thereof. 3.The oligopeptide of claim 1, wherein n=0 and y=0.
 4. The oligopeptide ofclaim 1, further comprising a flanking sequence.
 5. The oligopeptide ofclaim 1, further comprising a covalent linkage to all or a portion of amammalian tTGase.
 6. The oligopeptide of claim 5, wherein said mammaliantTGase is selected from the group consisting of a human, bovine, equine,and porcine tTGase.
 7. The oligopeptide of claim 6, wherein said tTGaseis covalently linked to said oligopeptide at a site of deamidation.
 8. Amethod for diagnosing Celiac Sprue in an individual, said methodcomprising detecting the presence of an antibody that specifically bindsan oligopeptide of claim 1 in a tissue, bodily fluid, or stool of saidindividual.
 9. A method for diagnosing Celiac Sprue in an individual,said method comprising determining whether an oligopeptide of claim 1stimulates agglutination of anti-gliadin antibodies, anti-tTGaseantibodies, or combinations thereof from said individual, andcorrelating an ability to stimulate agglutination with a positivediagnosis of Celiac Sprue.
 10. A method for diagnosing Celiac Sprue inan individual, said method comprising detecting the presence of anoligopeptide of claim 1 in a tissue, bodily fluid, or stool of saidindividual.
 11. The method of claim 10, wherein said detecting isaccomplished using an antibody that recognizes said oligopeptide or by acell line that proliferates in the presence of said oligopeptide.
 12. Apurified oligopeptide selected from the group consisting of (SEQ IDNO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF, a deamidated counterpartthereof, and a conservatively modified variant thereof.
 13. Theoligopeptide of claim 12, wherein at least one of the underlined Qresidues in (SEQ ID NO:12) LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF isdeamidated.
 14. The oligopeptide of claim 12, further comprising acovalent linkage to all or a portion of a mammalian tTGase.
 15. Theoligopeptide of claim 14, wherein said mammalian tTGase is selected fromthe group consisting of a human, bovine, equine, and porcine tTGase. 16.The oligopeptide of claim 15, wherein said tTGase is covalently linkedto said oligopeptide at a site of deamidation.
 17. A method fordiagnosing Celiac Sprue in an individual, said method comprisingdetecting the presence of an antibody against an oligopeptide of claim12 in a tissue, bodily fluid, or stool of said individual.
 18. A methodfor diagnosing Celiac Sprue in an individual, said method comprisingdetermining whether an oligopeptide of claim 12 stimulates agglutinationof anti-gliadin antibodies, anti-tTGase antibodies, or combinationsthereof from said individual, and correlating an ability to stimulateagglutination with a positive diagnosis of Celiac Sprue.
 19. A methodfor diagnosing Celiac Sprue in an individual, said method comprisingdetecting the presence of an oligopeptide of claim 12 in a tissue,bodily fluid, or stool of said individual.
 20. The method of claim 19,wherein said detecting is accomplished using an antibody that recognizessaid oligopeptide or by a cell line that proliferates in the presence ofsaid oligopeptide.
 21. The method of claim 8, wherein said tissue is amucosal tissue selected from the group consisting of oral, nasal, lung,and intestinal mucosal tissue.
 22. The method of claim 8, wherein saidbodily fluid is selected from the group consisting of blood, sputum,urine, phlegm, lymph, and tears.
 23. An antibody-producing cell linethat produces an antibody that binds specifically to an oligopeptide ofclaim
 1. 24. An antibody produced from the cell line of claim
 23. 25.The method of claim 8, wherein said individual has not consumed glutenfor an extended period of time.
 26. The method of claim 25, wherein saidextended period of time is selected from the group consisting of oneday, one week, one month, and one year prior to the performance of thediagnostic method.
 27. The method of claim 8, wherein said individualhas not had an endoscopy.
 28. The method of claim 8, wherein saidindividual is the subject of a therapy intended to treat Celiac Sprue oris in a clinical trial conducted to evaluate such a therapy.